The response of human leukocytes to chemoattractants generated at inflammatory sites is critical to their role in the innate immune response. Signaling from G protein-coupled chemoattractant receptors to the cellular cytoskeleton involves Rho GTPases and the downstream mediator, p21-activated kinase (PAK). We have identified a novel PAK molecular partner as GEF- H1, a Rho family guanine nucleotide exchange factor (GEF) associated with microtubules. GEF-H1 provides a potential link between Rho GTPase regulation of the actin-myosin cytoskeleton and microtubule dynamics. We will use molecular and genetic approaches to investigate the regulation and biology of PAK and GEF-H1. Critical questions remain about the spatial and temporal regulation of Rho GTPase function in the context of a polarized, chemotactically responding cell. We will apply a novel method of live cell analysis for detecting Rho GTPase activation (FLAIR) to relate the kinetics of Rho GTPase activation to chemotaxis response parameters, to changes in the cellular cytoskeleton, and to biochemical signaling events. The role(s) of PAK and its molecular partner, PIX, as mediators of Rac and Cdc42 in chemotaxis will be investigated, and their mechanisms of action determined using biochemical and genetic approaches. Our ability to achieve these goals relies on our development of FLAIR technology, as well as effective means to introduce protein components into leukocytes and model cell chemotaxis systems. The proposed studies should yield new insights into the regulation of cell motility and the inflammatory response of human leukocytes.